Protection of injured or infected tissue involves the coordinated delivery of migratory leukocytes. Thus, current research efforts aim to define the mechanisms that control movement of these cells into and out of specific tissues. Here, we will focus on monocytes because these cells play a central role in the orchestration and resolution of tissue inflammation, and thus represent attractive targets in therapy. Monocytes recruited to tissue are thought to originate exclusively from blood and bone marrow. However, recent work indicates that the resting spleen contains bona fide, undifferentiated monocytes that outnumber their blood equivalents. Splenic monocytes are important because they can be massively mobilized to inflammatory sites upon appropriate stimulation. These findings form the basis of our hypothesis that the spleen is a site for storage and rapid deployment of monocytes, and that splenic monocytes represent a significant resource that the body exploits to quantitatively control inflammation. At present, there is a need to clarify the spleen-associated mechanisms that control monocyte homeostasis and function. In a first aim, we will define important parameters that define the biology of monocytes in the resting spleen (e.g., cell turnover, location and organization, gene expression and behavior). These studies are necessary to uncover fundamental aspects of monocyte behavior and the role of the spleen as a monocyte storage organ. In a second aim, we will define mechanisms that are employed to activate and release splenic monocytes. Preliminary studies identified two instigators of inflammation with potent but distinct spleen-related effects;these compounds will be analyzed in detail. The project will use state-of-the-art readouts that are available in the laboratory, e.g., gene profiling of [unreadable]minimallytouched[unreadable] monocyte subsets and lineage descendants for comprehensive analysis in distinct tissues, and timelapse intravital cellular imaging to interrogate the behavior of monocytes directly in vivo. We have developed and/or validated the technology over the past three years, and will now apply it to answer the biological questions relevant to monocytes. Our experiments will compare two monocyte subsets (Ly-6Chi and Ly-6Clo) because they appear to play different roles in immunity, yet their relative contributions need clarification. In vivo imaging will use newly-generated Cx3cr1gfp/+ Ccr2rfp/+ mice since monocyte subsets in these mice can be followed separately. The findings should have broad applicability because monocytes participate actively in a plethora of life-threatening or debilitating diseases. The ultimate goal is the identification of new therapeutic options that control the mobilization and activation of monocytes, and thus enhance or suppress inflammation. The project will interact closely with local (Weissleder, Luster and Xavier at MGH;von Andrian at HMS) and outside (Charo at UCSF) immunology, imaging and systems biology groups.